Strains of Phaffia rhodozyma containing high levels of astaxanthin and low levels of 3-hydroxy-3&#39;,4&#39;-didehydro-β, Ψ-caroten-4-one (HDCO)

ABSTRACT

The present invention provides Phaffia rhodozyma strains having increased levels of astaxantin and a low percentage of 3-hydroxy-3&#39;,4&#39;-didehydro-β,ψ-caroten-4-one (HDCO). Such strains are obtained by a combination of mutagenesis and selection. The invention further provides a method for obtaining such strains. The invention also provides astaxanthin obtained from a mutagenized strain of Phaffia rhodozyma characterized in that it contains a decreased relative amount of HDCO.

FIELD OF THE INVENTION

This invention relates to the microbial production of astaxanthin.Specifically, yeast strains are disclosed with a high astaxanthinproduction level and a low level of3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-one (HDCO).

BACKGROUND OF THE INVENTION

Astaxanthin is a naturally occurring carotenoid which is responsible forthe red colour in salmon, trout, sea bream and other fishes. In naturalsurroundings this carotenoid is obtained with the feed of these animalsin the form of crustaceans and other astaxanthin-containing organisms.

Fish raised on fish-farms or in hatcheries are generally pale and lackthe skin and flesh colours of their naturally raised congeners, this isdue to a lack of dietary astaxanthin. The addition of astaxanthin to thefeed of the fishes is an appropriate way to overcome the colour problemof fish raised in non-natural surroundings.

Astaxanthin applied for this purpose can be obtained by semi-syntheticalmeans. For regulatory reasons and due to increasing pressure excercisedby consumers it may be favourable to use astaxanthin from naturalsources.

Natural sources of astaxanthin are krill and crawfish shells, algae,flowers and yeast. Astaxanthin extracted from krill and crawfish is veryexpensive. The yield of astaxanthin in algae is high but large-scaleproduction of algae is difficult. Therefore attention has turned to theuse of yeast, specifically Phaffia rhodozyma for the production ofastaxanthin.

The astaxanthin-containing yeast Phaffia rhodozyma was first isolated inthe early 1970s from exudates of deciduous trees in mountainous regionsof Japan and Alaska (Phaff et al. (1972) in Proceedings of the 4th IFS:Fermentation Technology Today, p. 759-774. Ed. G. Terui, Kyoto). Inwild-type Phaffia rhodozyma strains astaxanthin concentrations have beendetermined. Johnson and Lewis ((1979), J. Gen. Microbiol. 155 173-183)have found that the concentration of astaxanthin which is dependent onculture conditions varies considerably but never exceeds 650 μg per gyeast dry weight. When growing the yeast under different growthconditions the authors have found, after acetone extraction of thecarotenoids, that one of the main contaminants of astaxanthin is3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-one (HDCO).

Under aerobic conditions the amount of HDCO is about 0.5-1.5% of theamount of astaxanthin. Under microaerophilic conditions this amountincreases up to about 26%, (Johnson and Lewis cited above) this amountis too high.

The astaxanthin concentration of about 650 μg/g dry weight as reportedfor wild type Phaffia rhodozyma strains is much too low to make theprocess economically attractive. Therefore extensive studies have beenperformed to increase the yield of astaxanthin.

As indicated above Johnson and Lewis (1979) already describe a myriad ofdifferent culture conditions (pH, temperature, carbon source, oxygenpressure and light are but a number of factors) which influence theintracellular astaxanthin concentration in Phaffia. A more promisingapproach for obtaining higher astaxanthin concentrations is classicalmutagenesis. An and Johnson ((1990) Antonie van Leeuwenhoek 57: 191-203)report that N-methyl-N'-nitro-N -nitrosoguanidine (NTG) mutagenesis ofnatural Phaffia rhodozyma strains gave rise to strains with increasedcarotenoid contents, amounts of up to 1050 μg/g dry weight are reported.

The strains grown under different conditions described by An and Johnson(opt.cited) were found to contain from 10-15% of total carotenoids inthe form of HDCO (about 30% of the amount of astaxanthin). Also Lewis etal ((1990) Appl. Environm. Microbiol. 56: 2944-2945) have shown that theamount of HDCO as compared to astaxanthin increases due to mutagenesis.In their situation (Table 1) HDCO increased from 4 to 8%.

No biological effects of HDCO are known. In fact till now HDCO has onlybeen reported in Phaffia rhodozyma. High amounts of HDCO which seem tobe increasing due to mutagenesis are unacceptable as additions to animalfeed.

Therefore there is clearly a need for astaxanthin obtained from Phaffiawhich has a low HDCO content. The present invention provides such anastaxanthin.

SUMMARY OF THE INVENTION

The present invention provides Phaffia rhodozyma strains having a highastaxanthin content with a low percentage of HDCO.

The present invention also provides astaxanthin obtained frommutagenized Phaffia rhodozyma strains having a high astaxanthin contentwith a low percentage of HDCO.

The present invention further provides a method for obtaining Phaffiarhodozyma strains having a high astaxanthin content with a lowpercentage of HDCO.

In reverse this method can be employed for obtaining a Phaffia rhodozymastrain having a decreased level of astaxanthin and a low percentage ofHDCO.

DETAILED DESCRIPTION OF THE INVENTION

Phaffia rhodozyma CBS 6938 was obtained from the Centraal Bureau voorSchimmelcultures (Baarn, The Netherlands).

In general terms the present invention discloses Phaffia rhodozymastrains having a high astaxanthin level and a relatively low percentageof HDCO. Said strains are obtained by a method comprising the followingsteps;

mutagenizing a Phaffia rhodozyma strain,

visually selecting a strain with increased colour intensity or visuallyselecting (intensively) coloured orange colonies with high astaxanthinand low HDCO content compared to the astaxanthin content, from apopulation of red colonies (with high astaxanthin and a high HDCOcontent relative to the amount of astaxanthin),

growing the selected strain on a fluid medium,

extracting the carotenoids from the cells,

determining the amounts of carotenoids,

selecting a strain with an increased level of astaxanthin and a lowlevel of 3-hydroxy-3',4'-didehydro-β,ψ-caroten -4-one.

Mutagenesis is performed on the Phaffia rhodozyma strains in a number ofways for example by UV irradiation, or by treatment with certainmutagenic substances e.g. ethyl methane sulphonate,N-methyl-N'-nitro-N-nitrosoguanidine or other nucleotide base analogues.

Mutated strains are selected on the basis of colour intensity and/ordifference in colour and the carotenoids were extracted for example byacetone extraction.

Selection on the basis of colour difference is performed as follows

Appropriate dilutions of a mutagenized cell suspension were plated outon agar plates containing synthetic medium. The dilution was chosen insuch a way that 400-450 colonies grew on the surface of a 3,6 inchplate. After 4-6 weeks of incubation at 20° C. in the dark the orangecolonies were visually selected (colonies containing more than 10percent HDCO were supposed to be red) and were used for shake flaskexperiments. A 100 ml shake flask containing 25 ml synthetic medium wasinoculated with a strain isolated as described above and incubatedduring 3 days (20° C., 250 rpm). Hereafter, 1 ml of the cell suspensionwas used to inoculate 100 ml synthetic medium in a 500 ml shake flaskwith baffle and incubation occured for 4 days at 20° C., 250 rpm. Afterthis, the amount of astaxanthin, HDCO and dry weight were determined.

The amount of specific carotenoids was determined by suitablechromatographic techniques such as thin-layer chromatography, or HPLC.

To obtain strains with a higher astaxanthin content repeatedmutagenization/selection steps are performed.

Although generally it was found that the relative amount of HDCOincreased with an increasing amount of astaxanthin both in theliterature and in our experiments, strains have now been found with ahigh astaxanthin content and with a low percentage of HDCO.

A high level of astaxanthin means that the amount is increased ascompared with the wild type strain e.g. above about 650 μg/g dry weight(as shown in Johnson and Lewis. J. Gen. Microbiol. (1979) 115 178, Table3). Preferably the amount is above 2000 μg/g dry weight, more preferablyabove 8000 μg/g dry weight.

The amount of HDCO which in the wild type strain is of the order of0.5-1.0% relative to the amount of astaxanthin rapidly increases uponmutagenesis to levels of up to 35% relative to the astaxanthin level. Alow percentage of HDCO is defined as lower than 10% HDCO, preferablyless than 5% HDCO, more preferably less than 2% relative to astaxanthinor even lower than 1% HDCO whereby the astaxanthin is extracted from astrain containing an amount of astaxanthin higher than 650 μg/g dryweight.

The present invention provides strains which combine a high level ofastaxanthin with a low percentage of HDCO. Preferred strains are thosewhich have a level of astaxanthin above 1500 μg/g dry weight and apercentage of HDCO lower than 3% of the astaxanthin level.

To obtain Phaffia strains combining these characteristics it is possibleto mutagenize a strain which has already been selected for highastaxanthin production, and an example of this is provided.

However, it is also possible to start with a wild type strain and firstselect for a decrease in the HDCO percentage and subsequently mutagenizefor an increased level of astaxanthin (keeping the HDCO percentage low).

It is also possible to obtain strains having the desired characteristicsof high astaxanthin production with low HDCO level, by using protoplastfusion. The combination of protoplasts obtained from cells with highHDCO and protoplasts obtained from cells with low HDCO level results incells having the desired characteristics.

The astaxanthin isolated or contained in the cells can be formulated inanimal feed or food. Use of this food or feed will give rise to thedesired colour for example in salmonids, sea bream and eggs. The presentinvention thus provides an animal feed or food comprising yeast cells oryeast cell parts containing astaxanthin in an amount of at least 650μg/g dry yeast weight and a 3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-oneconcentration of less than 10% HDCO, preferably less than 5% HDCO, morepreferably less than 2% HDCO or even lower than 1% HDCO relative to theastaxanthin level in the cells.

The method can also be employed to obtain strains having a lowastaxanthin level and a low percentage of HDCO.

The following examples are only given to illustrate the invention andnot to limit the invention in any way. It is immediately clear to aperson skilled in the art that other mutagens and other Phaffia strainscan be used to obtain the same or similar results.

Experimental

Media

Biomalt medium

122 gram MEX 11 (Diastatische Producten B.V. Leiden-Holland) wasdissolved in 1 liter demineralized water, the pH was set at 6.4 and thesolution was sterilized for 20 minutes at 120° C. For the preparation ofBiomalt agar medium agar was added to a final concentration of 1.0%(w/v).

Potatodextrose (CM 11-2) agar medium

    ______________________________________                                        Potatodextrose (Difco)                                                                         39           g                                               demineralized water                                                                            1000         ml                                              agar             10           g                                               pH before autoclaving                                                                          7.4                                                          ______________________________________                                    

Sterilization is performed by heating for 20 minutes at 120° C.

Synthetic medium

    ______________________________________                                                        Concentration                                                 medium component                                                                              (g/l)                                                         ______________________________________                                        KH-phtalate     20                                                            pH              5.6                                                           NaCl            0.06                                                          MgSO.sub.4      0.88                                                          CaCl.sub.2      0.20                                                          H.sub.2 SO.sub.4                                                                              0.071                                                         NH.sub.4 Cl     4.83                                                          KH.sub.2 PO.sub.4                                                                             1                                                             citric acid     0.015                                                         (NH.sub.4).sub.2 Fe (SO.sub.4).sub.2                                                          0.027                                                         ZnSO.sub.4      0.005                                                         CuSO.sub.4      0.0075                                                        MnSO.sub.4      0.0006                                                        H.sub.3 BO.sub.3                                                                              0.0006                                                        Na-molybdate    0.0006                                                        KI              0.00015                                                       Myo-inositol    0.059                                                         nicotinic acid  0.003                                                         Ca-D-panthotenate                                                                             0.003                                                         vitamin B.sub.1 0.003                                                         p-aminobenzoate 0.002                                                         vitamin B.sub.6 0.0003                                                        biotin          0.00001                                                       glucose         33                                                            ______________________________________                                    

Sterilization was for 30 minutes at 110° C.

Maintenance of cultures

Cultures of Phaffia rhodozyma are stored in two ways:

a. Slants containing biomalt agar medium were prepared from Phaffiarhodozyma CBS 6938 and the mutants derived thereof. These slants areincubated 1 week at 20° C. and subsequently frozen at -20° C.

b. Vials are prepared from cultures which are grown in 100 ml shakeflasks (with baffle) containing 25 ml synthetic medium (20° C., 250rpm). Cultures are centrifuged and suspended in demineralized water(filtered in a milli q filter system from Millipore). containing 10%(v/v) glycerol, the vials are frozen in liquid nitrogen and stored at-80° C.

High Performance Liquid Chromatography

Extraction procedure

A sample of 2.5 g culture was pipetted into a closable 15 ml glasscentrifuge tube and centrifuged (3200× g, 25 minutes, 15° C.). Thesupernatant was removed. 1.5 ml cold aceton (5° C.) and 5 g glass beads(3 mm diameter) were added and mixed by a vortex. The centrifuge tubeswere mixed on a Vibrax™ mixer (IKA Model Vibrax VXR) and the mixer wasset at maximal speed during 60 minutes.

Hereafter, acetone (20° C.) was added to a final suspension volume of 10ml (without the glass beads). The suspension was homogenized andcentrifuged (3200× g, 5 minutes, 20° C.). The supernatant was used forHPLC analysis.

Equipment

Columns: Novapack™ C 18 Catridge (5×100 mm) (Waters Associates, Milford,Massachusetts)

Detector: Waters 486 Tunable Absorbance Detector

Control and

Integration: Integration was performed with software "Maxima package"commercially available from Waters Instruments.

Autosampler: Waters 712 Wisp with cool system.

Solvents

Bisethyl hexylphosphate (16 g/l) and formic acid (40 ml/l) in

acetonitril.

The solvents were HPLC grade.

Operation

Flow: 1 ml/min.

Detector: 471 nm

Temperature: 18° C.

Injection volume: 20 μl

Astaxanthin standard

A purified astaxanthin preparation (Gist-brocades, R&D department CMA,Delft, The Netherlands) was used as a standard. Briefly, purificationwas performed as follows; Astaxanthin in Phaffia-oil (Aquaculture 20123-134 (1980)), is treated with hexane. After filtration and washingthe crystalline product is dissolved in methylene chloride (MTC),methanol is added (in a ratio of 4:3 with respect to MTC) and themixture is concentrated under vacuum. The crude astaxanthin obtained isdissolved in chloroform and chromatographed over silica gel (withtoluene/acetone 4:1). The dried product is again crystallized from amixture of MTC and methanol, optionally this is repeated several times.As far as possible the procedure is performed protected from light.Further characterization of the product is by HPLC.

The astaxanthin content was expressed as mg astaxanthin/kg broth. Theamount of 3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-one was calculated inthe same way as astaxanthin. During the calculations it was assumed thatastaxanthin has the same extinction coefficient as 3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-one and therefore the same astaxanthinstandard sample was used for the calculations. The amount of3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-one was expressed in mg3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-one/kg broth.

Dry weight measurement

Plastic 12 ml centrifuge tubes (Greiner) were dried in an incubator for24 hours (105° C.). The tubes were cooled down in an excicator over drysilicagel and thereafter the weight (A) was determined in four decimalson a Mettler AE200 balance. The centrifuge tubes were filled with about10 ml cell suspension and the weight (B) was determined as describedabove. The cell suspensions were centrifuged at room temperature (10minutes, 4000 rpm) and the supernatant was decanted. The centrifugetubes plus pellets were dried in an incubator (24 hours, 105° C.).Afterwards the tubes were cooled down in the excicator and the weightwas measured as described above (C).

All dry weight measurements were performed in duplo.

Calculation: dry weight (g/kg)={(C-A)/(B-A)}*1000 and A,B,C wereexpressed in grams.

Chemicals

The standard samples of astaxanthin needed for the HPLC measurementswere prepared at Gist-brocades (CMA department). All other chemicalsused in these experiments are commercially available and of analyticalgrade.

Mutagenesis

Strain improvement was achieved by classical mutation. Mutations wereinduced by the use of NTG (N-methyl-N'-nitro-N -nitrosoguanine), EMS(ethylmethanesulphonate) or ultraviolet irradiation and a survival valueof 10% was considered to induce sufficient mutations. Appropriatedilutions of cell-suspensions were made on agar plates and afterincubation the colour intensity of the red colonies were compared by eyeand the colonies showing the highest red colour intensity were isolatedand used for shake flask experiments.

EXAMPLE 1

A strain of Phaffia rhodozyma with high astaxanthin and high3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-one content, Phaffia rhodozymaPF 11-3, was obtained through repeated rounds of mutagenesis andselection. This strain was used to isolate strains with low3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-one content. A 100 ml culturewas grown on biomalt medium in a 500 ml flask (with baffle) for 72 hoursin a shaking incubator (250 rpm, 20° C.) The culture was centrifuged(4000 rpm, 10 minutes) and suspended in 9 ml Tris-buffer (100 mM, pH8.0). The cell-density of the suspension was set at 10⁸ /ml.Subsequently, 1 ml 10 mM NTG in 100 mM Na-acetate buffer pH 4.3 wasadded to 10 ml of the cell suspension. At appropriate time intervalssamples were drawn and a part of the sample was diluted in physiologicalsalts medium and plated out on potato-dextrose agar for a survival test.Another part of the sample was frozen in 10% glycerol. Afterdetermination of the 10% survival the involved sample was plated out onpotato-dextrose agar and incubated one week at 20° C. Colonies with themost intensive red colour were selected by eye and tested in a 100 mlshake flask (with baffle) containing 25 ml synthetic medium (250 rpm,20° C.). After 72 hours 1 ml of this culture was used to inoculate 100ml of the same medium in a 500 ml shake flask (with baffle) andincubated under the same conditions as is described for the preculture.After 98 hours 2.5 g culture was used to prepare a sample for HPLC and10 ml was used for a dry weight measurement. Strain PF 11-12 wasselected after mutagenesis of the parent strain PF 11-3 and theastaxanthin and 3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-one content wasestimated by HPLC. In Table 1 the values of astaxanthin and3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-one content of these strains arepresented and compared with the wild type strain Phaffia rhodozyma CBS6938.

                  TABLE 1                                                         ______________________________________                                                                 3-hydroxy-3',4'-                                                    Astaxanthin                                                                             didehydroxy-β,ψ-                                           content   caroten-4-one                                        Strain         (μg/g DWT)                                                                           ratio*                                               ______________________________________                                        Phaffia rhodozyma                                                                            220       0.20                                                 CBS 6938                                                                      Phaffia rhodozyma                                                                            1280      0.22                                                 PF 11-3                                                                       Phaffia rhodozyma                                                                            1670      0.02                                                 PF 11-12                                                                      Phaffia rhodozyma                                                                            n.d.      0.17                                                 CBS 215.88                                                                    Phaffia rhodozyma                                                                            n.d.      0.37                                                 CBS 224.87                                                                    Phaffia rhodozyma                                                                            n.d.      0.16                                                 CBS 225.87                                                                    ______________________________________                                    

The Phaffia rhodozyma strain CBS 215.88, CBS 224.87 and CBS 225.87 andthe method by which they have been obtained is described in WO 88/08025.Briefly, Phaffia rhodozyma ATCC 24261 was mutagenized with EMS to obtainstrain CBS 224.87. Strain CBS 224.87 was subsequently mutagenized withNTG to obtain CBS 225.87. Finally, strain CBS 215.88 is a reisolate fromstrain CBS 225.87. It is observed that these mutant strains contain arelatively high level of HDCO.

It is noted that when the mutant strain PF 11-12 is compared with theparent strain PF 11-3, PF 11-12 contains an increased amount ofastaxanthin while the 3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-onecontent decreased a factor 10. PF 11-12 was deposited at Centraal Bureauvoor Schimmelcultures (Baarn, The Netherlands) on 16 December 1991 undernumber CBS 797.91.

EXAMPLE 2

A culture of Phaffia rhodozyma PF 11-12 was treated as described inExample 1, with the alteration that 0.4 ml EMS (Merck) was used as amutagen instead of 1 ml 10 mM NTG. The selection was carried out asdescribed in Example 1. Strain PF 11-18 was selected from the parentstrain PF 11-12 and in Table 2 the values of astaxanthin and3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-one contents are presented:

                  TABLE 2                                                         ______________________________________                                                       Astaxanthin                                                                             3-hydroxy-3',4'-                                                    content   didehydro-β,ψ-                              Strain         (μg/g DWT)                                                                           caroten-4-one ratio*                                 ______________________________________                                        Phaffia rhodozyma                                                                            220       0.20                                                 CBS 6938                                                                      Phaffia rhodozyma                                                                            1670      0.02                                                 PF 11-12                                                                      Phaffia rhodozyma                                                                            1800      0.03                                                 PF 11-18                                                                      ______________________________________                                         *as in Table 1                                                           

In this example it is shown that mutants with higher astaxanthin contentcan be isolated from parent strains containing low3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-one (PF 11-12), while the3-hydroxy-3',4'-didehydro-β,ψ-caroten -4-one content of the mutantstrain (PF 11-18) remains low.

EXAMPLE 3

A culture of Phaffia rhodozyma PF 11-18 was treated as described inExample 1 and 1 ml 10 mM NTG was used. The selection was carried out asdescribed in Example 1. Strain PF 11-30 was selected from the parentstrain PF 11-18. In Table 3 the values of astaxanthin and3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-one contents are presented:

                  TABLE 3                                                         ______________________________________                                                       Astaxanthin                                                                             3-hydroxy-3',4'-                                                    content   didehydro-β,ψ-                              Strain         (μg/g DWT)                                                                           caroten-4-one ratio*                                 ______________________________________                                        Phaffia rhodozyma                                                                            220       0.20                                                 CBS 6938                                                                      Phaffia rhodozyma                                                                            1800      0.03                                                 PF 11-18                                                                      Phaffia rhodozyma                                                                            1960      0.02                                                 PF 11-30                                                                      ______________________________________                                         *as in Table 1                                                           

In this example it is shown that mutants with higher astaxanthin contentcan be isolated from parent strains containing low3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-one (PF 11-18), while the3-hydroxy-3',4'-didehydro-β,ψ-caroten -4-one content of the mutantstrain (PF 11-30) remains low.

EXAMPLE 4

A strain of Phaffia rhodozyma , namely PF 11-36 obtained aftermutagenesis of strain PF 11-12, containing a high level3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-one content relative toastaxanthin, was used to isolate strains with a low3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-one content ratio. A 100 mlculture was grown on synthetic medium in a 500 ml flask (with baffle)for 65 hours in a shaking incubator (250 rpm, 20° C.). The culture wascentrifuged (4000 rpm, 10 minutes) and suspended in 25 ml Tris buffer(100 mM, pH 7.5). The cell density of the suspension was set at 10⁸ /ml.Subsequently, 1 ml 10 mM NTG in 100 mM acetate buffer pH 4.3 was addedto 10 ml of the cell suspension. At appropiate time intervals sampleswere drawn and were diluted into 0.1 M Tris buffer pH 7.5. The cellswere washed three times in physiological salts medium and plated out onbiomalt agar medium to estimate the survival. A part of the dilution wasfrozen in 10% glycerol (-80° C.). After determination of the samplecontaining 10% survival, appropriate dilutions of the frozen sample wereplated out on the synthetic medium agar (100-500) colonies/3,6 inchplate). After 4-6 weeks incubation (20° C.) the orange coloured colonieswere visually selected and tested in shake flask experiments asdescribed above. In table 4 the astaxanthin contents and3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-one ratio's of the variousisolated strains of Phaffia rhodozyma PF 11-36 are presented.

These results show clearly that by selection on colour, mutants can beselected with astaxanthin levels comperable with that of the parentstrain. These strains contain high levels of astaxanthin but the3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-one ration has been stronglyreduced in relation to the amount of astaxanthin.

                  TABLE 4                                                         ______________________________________                                        Astaxanthine contents and 3-hydroxy-3',4'-didehydro-                          β,ψ-caroten-4-one ratio's of the various isolated strains of         Phaffia rhodozyma PF 11-36.                                                                            3-hydroxy-3',4'-                                                    Astaxanthine                                                   didehydro-β,                                                                            content                                                        caroten-                                                                      Strain         (μg/g DWT)                                                                           4-one ratio*                                         ______________________________________                                        Phaffia rhodozyma                                                                            2303      0.14                                                 PF 11-36                                                                      Phaffia rhodozyma                                                                            1545      0.05                                                 PF 11-36-13                                                                   Phaffia rhodozyma                                                                            542       0.05                                                 PF 11-36-15                                                                   Phaffia rhodozyma                                                                            1104      0.07                                                 PF 11-36-17                                                                   Phaffia rhodozyma                                                                            916       0.08                                                 PF 11-36-27                                                                   Phaffia rhodozyma                                                                            643       0.06                                                 PF 11-36-47                                                                   Phaffia rhodozyma                                                                            852       0.05                                                 PF 11-36-113                                                                  Phaffia rhodozyma                                                                            899       0.07                                                 PF 11-36-123                                                                  Phaffia rhodozyma                                                                            1795      0.07                                                 PF 11-36-240                                                                  Phaffia rhodozyma                                                                            521       0.06                                                 PF 11-36-263                                                                  Phaffia rhodozyma                                                                            1200      0.09                                                 PF 11-36-422                                                                  Phaffia rhodozyma                                                                            2205      0.02                                                 PF 11-36-489                                                                  Phaffia rhodozyma                                                                            2180      0.07                                                 PF 11-36-500                                                                  Phaffia rhodozyma                                                                            931       0.04                                                 PF 11-36-653                                                                  Phaffia rhodozyma                                                                            970       0.03                                                 PF 11-36-672                                                                  Phaffia rhodozyma                                                                            1580      0.01                                                 PF 11-36-678                                                                  Phaffia rhodozyma                                                                            1408      0.03                                                 PF 11-36-696                                                                  ______________________________________                                         *the 3hydroxy-3',4didehydro-β,caroten-4-one ratio is defined as the      ratio of 3hydroxy-3',4didehydro-β,caroten-4-one content and              astaxanthin content plus the 3hydroxy-3',4didehydro-β,caroten-4-one      content.                                                                 

We claim:
 1. A Phaffia rhodozyma strain having an intracellularconcentration of more than 650 μg astaxanthin per g dry weight and a3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-one (HDCO) percentage relativeto the amount of astaxanthin of less than 5% by weight.
 2. The P.rhodozyma strain of claim 1 which has an intracellular astaxanthinconcentration of more than 1,500 μg/g of dry weight.
 3. The P. rhodozymastrain of claim 2 which has an intracellular astaxanthin concentrationof more than 2000 μg per g of dry weight.
 4. The P. rhodozyma strain ofclaim 1 wherein the percentage of HDCO relative to astaxanthin is lessthan 2% by weight.
 5. The P. rhodozyma strain of claim 4 wherein thepercentage of HDCO relative to astaxanthin is less than 1% by weight. 6.The P. rhodozyma strain of claim 1 which has an intracellularastaxanthin content of more than 1500 μg/g dry weight and a percentageof HDCO less than 3% of astaxanthin by weight.
 7. The P. rhodozymastrain of claim 6 in which the percentage of HDCO is less than 1% ofastaxanthin by weight.
 8. A method for modifying the relativeastaxanthin and HDCO levels of a Phaffia rhodozyma strain, which methodcomprises:culturing a Phaffia rhodozyma strain having an astaxanthincontent of more than 650 μg per g dry weight and a level of3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-one (HDCO) of greater than 5% byweight relative to the amount of astaxanthin, mutating the Phaffiarhodozyma strain, visually selecting colonies having a relatively moreintense orange color than the other colonies, growing the selectedorange colonies on a fluid medium, extracting the carotenoids from thecells of said colonies, determining the amounts of astaxanthin and HDCOin the carotenoid extracts, and selecting a strain having an astaxanthincontent of more than 650 μg per g dry weight and a level of3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-one (HDCO) of less than 5% byweight relative to the amount of astaxanthin.
 9. A method for modifyingthe astaxanthin and HDCO levels of a Phaffia rhodozyma strain, whichmethod comprises:culturing a parent Phaffia rhodozyma strain having anastaxanthin content of more than 650 μg per g dry weight and a level of3-hydroxy-3',4'-didehydro-β,ψ-caroten-4-one (HDCO) of greater than 5% byweight relative to the amount of astaxanthin, mutating the Phaffiarhodozyma strain, visually selecting colonies having a relatively moreintense orange color than the other colonies, growing the selectedorange colonies on a fluid medium, extracting the carotenoids from thecells of said colonies, determining the amounts of astaxanthin and HDCOin the carotenoid extracts, and selecting a strain having an astaxanthincontent less than that of the parent strain and a level of HDCO of lessthan 5% by weight relative to the amount of astaxanthin.